Combining a biomechanical approach and two unique animal model systems that mimic two different aspects of brain arteriovenous malformations (AVMs), our new proposal will focus on determining the consequences of MMP inhibition at the cellular and structural levels. We will demonstrate that the abnormal levels of matrix metalloproteinases (MMPs) in AVMs and resultant vascular instability are due to a combination of two mechanisms[unreadable]high blood flow rate induced MMP expression in inflammatory and vascular cells and AVM vascular cell's intrinsic property to abnormally express high levels of MMPs. When MMPs, which originated from these two underlying mechanisms, are inhibited pharmacologically or abolished genetically, it results in restoration of vascular stability at the structural and cellular levels. Specific Aim 1: Roles of high blood flow induced-MMPs and inflammation on structural integrity of blood vessels. We hypothesize that high blood flow rate will increase MMP levels in the vascular wall and cause vascular instability. Structural instability of blood vessels caused by high blood flow induced-MMP expression / activation can be reversed by inhibition MMPs. Specific Aim 2: Roles of abnormal expression of MMP-9 from AVM vascular cells on maintenance of pro-angiogenic phenotypes. We hypothesize that expression of abnormally high levels of MMP-9 by AVM vascular cells is their intrinsic property and that abnormally high levels of MMP-9 result in vascular instability. AVM tissues implanted in the brain and under the kidney capsule retain intrinsic pro-angiogenic phenotype[unreadable]high levels of MMP-9 and MMP-9 associated changes in other angiogenic factors[unreadable]while control tissues (superficial temporal artery and normal brain cortex samples) retain angiogenically quiescent phenotype. MMP inhibition changes the implanted AVM tissues from pro-angiogenic phenotype to quiescent and stable phenotype by decreasing the release and availability of other angiogenic factors such as VEGF. Significance: This project will elucidate two potential mechanisms for the abnormal expression of MMP-9 in AVMs. Furthermore, we will demonstrate that MMP inhibition can modify angiogenic phenotype of AVM tissues and restore structural stability of blood vessels. Findings from this study will be a basis for development of MMP inhibition treatment to restore vascular stability of AVMs in patients and prevent future hemorrhage. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page 225 Continuation Format Page Principal Investigator/Program Director (Last. First, Middle): PI: Hashimoto, T / Director: Young, WL. Introduction We would like to thank the study section for many positive and constructive comments that enabled us to refine and focus our project. The original proposal for Project 2 submitted in May 2007 received a score of 1.9. The committee pointed out that this proposal is "a good project with interesting ideas and using innovative molecular tools" and that "the pilot data is relatively strong," The committee also noted "the interactions with other projects and cores are very good". Four main suggestions for improvement from the committee included (1) the need to more carefully define hemodynamic conditions in the flow augmentation models, (2) the need to be careful with difficult controls in the implantation models, (3) the need to carefully define how inflammatory baselines might alter outcomes in the SCID mice, and (4) the need to pay some attention to consequences of long term MMP suppression. In addition, the committee noted some question regarding the appropriateness of the proposed models. We will address these five points raised by the committee, followed by specific critiques from each reviewer. As shown in this paragraph, major changes in the text are denoted by a double line in the right margin of the text and identified by a bracketed marker (e.g., <RESPONSE 1>). <Main criticisms from the committee> 1. More detailed characterizations of hemodynamic conditions in the surgical augmentation models To carefully define local and systemic hemodynamic conditions in flow-augmentation models, we performed new preliminary experiments. First, we established a blood pressure measurement method using a tail cuff system, by validating it against the intra-arterial catheter method that is considered to be a gold standard for blood pressure measurement. Using the tail cuff method, we found that neither unilateral carotid ligation nor sham surgery causes any significant changes in blood pressure in mice, consistent with previous finding by others.63 Along with our blood flow data, these data indicate that an increase in blood flow, not in blood pressure, is a major triggering factor for outward vascular remodeling in our model. We will follow blood pressure using a tail-cuff method in both the wild type and knock out mice during the experiments. Second, we performed preliminary studies to characterize differences in the flow-augmented vascular remodeling among different strains of wild type mice and knockout mice. There were baseline differences in the vascular diameter among different strains of mice. However, there was no difference in the baseline vascular diameter between the knockout mice and corresponding wild type mice. Although the degree of outward vascular remodeling was slightly different among different strains of mice, outward vascular remodeling was found to be a universal phenomenon. These findings emphasize (1) the need for baseline measurements of local and systemic hemodynamics and vascular diameter, and (2) the necessity for appropriate control groups to control for baseline differences among strains. Third, we have established another method to characterize blood flow in the mouse common carotid artery using an ultrasonic transit probe (Transonic) as presented in Preliminary Studies. This technique allows us to measure acute changes in the blood flow in more details than the pulse wave Doppler-based technique. We are currently applying the ultrasonic transit probe to the mouse basilar artery. We verified that there is an increase in basilar artery diameter after the common carotid ligation in mice. This finding provides indirect evidence that common carotid ligation causes outward remodeling of the basilar artery (a true intracranial artery) presumably mediated by the increase in the blood flow. As noted above, we verified that unilateral common carotid ligation does not change the blood pressure acutely or chronically. 2. Control tissues in implantation model The reviewer raised a concern regarding the use of structurally normal cerebral cortex tissues obtained during epilepsy surgery as a control in the implantation model. We have been using the structurally normal cerebral cortex tissues as control tissues, because these tissues are highly vascularized and have high density of endothelial cells. In our previous report,45 we showed that endothelial cells in these control samples exhibited normal morphology and were angiogenically quiescent and were indistinguishable from normal brain cortex obtained from the autopsy samples. Nevertheless, we agree that the microvasculature in cortex may be structurally and functionally different from larger blood vessels in the AVM nidus. As the reviewers acknowledged, normal human intracranial blood vessels with the similar diameter and structure are not available. However, they made a constructive suggestion of using normal artery from the same human. Therefore, in addition to the normal cerebral cortex samples, we will use the superficial temporal artery samples harvested during the surgical exposure. We currently have collected and snap frozen ten specimens for other purposes. We estimate that at least five superficial temporal artery samples per year will be available PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page 226 Continuation Format Page